Compared to standard methods based on monoclonal antibodies, the development of new aptamers (e.g., using Systematic Evolution of Ligands by Exponential Enrichment [SELEX]) is faster, simpler, more robust, and yields aptamers that can bind selectively and with excellent sensitivity to a wide variety of targets, including small organic molecules, proteins, antibodies, and even cells. Coupled with an appropriate transduction method, aptamers could be the basis of a universal multiplexed detection strategy capable of the simultaneous detection of many different classes of analytes in the same sample.
The advantageous properties of aptamers as a molecular recognition element have inspired the development of biosensors capable of detecting aptamer-ligand binding events. Significant progress has been made in the development of colorimetric, electrochemical, fluorescence, and mass-sensitive strategies. However, these detection methods are fundamentally limited for multiplexed applications. When aptamer-ligand binding occurs in the bulk phase (e.g., as used in nanoparticle colorimetric and label-free fluorescence assays) a characteristic detection signal for each target species is required (e.g., fluorescence emission wavelength), placing a finite constraint on multiplexing capacity. Other strategies, such as mass-sensitive and electrochemical detection, confine the aptamer-ligand binding to an interface and thus have the potential for site-dependent multiplexing. Unfortunately, signal transduction in many of these approaches is highly non-specific (e.g., surface adsorption or localization of redox species) and the presence of even small amounts of interfering species will produce a false response.
Accordingly, there is a need for a universally multiplexed aptasensor. In particular, the transduction element should ideally be label-free and respond specifically to aptamer-ligand binding.
Another area of current interest is understanding how to control bilayer fusion. Such an understanding is fundamentally and technologically important for designing synthetic gene transfer agents, drug delivery strategies, studying biological systems, and developing diagnostic assays. In particular, in vivo biomimetic strategies for studying receptor-mediated fusion have played a major role in the advancement of this field. Since Rothman and coworkers first demonstrated that SNARE proteins were the minimum machinery required for inducing membrane fusion, they have been widely accepted as the most efficient fusogenic receptors. Their biological origin and prevalence in cellular membranes have inspired exploration of the mechanisms that allow SNARE proteins to work with such high efficacy. A common motif has been found among SNARE receptors that involve a bundle of four alpha-helices that associate upon recognition. The configuration of this quaternary structure induced strain to the associated lipid bilayers, initiating the fusogenic process. Several synthetic approaches that mimic this structural motif have been developed using peptides, model proteins, small molecules, and DNA in an effort to achieve efficient recognition, bilayer disruption, and content transport in vivo.
In particular, DNA hybridization mediated fusion shows promise as a reductionist system both for studying fusion mechanics and as a bio-sensing strategy. Studies have shown that DNA can be anchored to lipid bilayers using DNA-lipid conjugates or sterol tethered DNA. Unilamellar liposomes can therefore be prepared with such tethered oligonucleotides. When two liposomes prepared with different but complementary oligonucleotides were combined, lipid mixing assays revealed bilayer fusion. A critical requirement in these assays was that membrane anchors on complementary DNA strands were necessarily on opposite ends of the DNA (i.e. 5′ and 3′ ends). In this configuration, DNA hybridization mimicked the configuration of the four helix bundle in SNARE receptors, brought the two bilayers into close proximity, strained the bilayer structure, and consequently induced efficient lipid mixing and content transport. In the alternative situation where the tethers were on the same end of the DNA, the liposomes were observed to aggregate but no lipid mixing or content transport occurred, presumably due to a lack of bilayer-bilayer proximity and strain.
Studying receptor-mediated fusion in dispersed liposomes is convenient for proof-of-concept studies but has limited capacity for advancing related technologies. Alternatively, receptor-mediated fusion with planar interfaces, and in particular supported lipid bilayers, has been used for quantitative high throughput studies that elucidate cellular mechanisms related to drug discovery, medical diagnostics, and biosensor development. Supported lipid bilayers can be fabricated as spatially addressed microarrays capable of high throughput screening and have demonstrated value as a tool for studying a range of biochemical processes. Despite their success as model systems, supported lipid bilayers possess complicating factors such as interfering effects associated with the underlying solid substrate and the necessity of complex and expensive analytical instrumentation. Thus, substrates that address some of these drawbacks have significant value toward a better understanding of liposome fusion from a fundamental and technological perspective.